1. Field of the Invention
This invention relates to the field of molecular biology, more particularly to methods and compositions involving nucleic acids, and still more particularly to methods and compositions for amplifying nucleic acids, e.g., genomic DNA, using nicking agents.
2. Description of the Related Art
A number of methods have been developed for whole genome amplification. Most of these methods involve the use of random or partially random primers to amplify the entire genome of an organism in a PCR reaction (see, e.g., Kuukasjarvi et al., Genes, Chromosomes and Cancer 18: 94–101 (1997); Telenius et al., Genomics 13: 718–25, 1992; Zhang et al., Proc. Natl. Acad. Sc. USA 89: 5847–51, 1992; Cheung et al., Proc. Natl. Acad. Sci. 93: 14676–79, 1996; Barrett et al., Nucleic Acids Res. 23: 3488–92; Klein et al., Proc. Natl. Acad. Sci. USA 96: 4494–9, 1999; Sun et al., Nucleic Acids Res. 23: 3034–40, 1995; Larsen et al., Cytometry 44: 317–325, 2001; and Barbaux et al., J. Mol. Med. 79: 329–32, 2001). This technique relies on having a sufficient number of primers of random or partially random sequences so that pairs of primers hybridize throughout the genomic DNA at moderate intervals. Extension from the 3′ termini of the primers produces strands to which another primer anneals. By subjecting the genomic DNA to multiple amplification cycles, the genomic sequences are amplified. Since this technique relies on PCR, it has the disadvantage that the amplification reaction must be carried out under cycles of different temperatures to achieve cycles of denaturation and re-annealing. Such cycles of denaturation and re-annealing are disadvantageous for many reasons, e.g., they may cause gene shuffling artifacts.
An alternative method for whole genome amplification is known as whole genome strand displacement amplification. This technique involves hybridization of random or partially random primers to a target genomic DNA and replication of the target sequence primed by the hybridized primers so that replication of the target sequence results in replicated strands complementary to the target sequence (see, e.g., U.S. Pat. Nos. 6,124,120 and 6,280,949). During replication, the growing replicated strands displace other replicated strands from the target sequence (or from another replicated strand) via strand displacement replication. Displacement of replicated strands by other replicated strands allows the amplification of a target sequence or portions thereof. Although this technique may be carried out under an isothermal condition, it requires the use of multiple primers.
There is a long felt need in the art for a simpler and more efficient method to amplify a whole genome. The present invention fulfills this and related needs as described below. In contrast to previously known techniques for whole genome amplification, the present invention provides a method for nucleic acid amplification that does not require the use of an external oligonucleotide primer. In addition, the present invention can be carried out under an isothermal condition, in other words isothermally, thus avoiding the expenses associated with the equipment for providing cycles of different temperatures and potential re-annealing or gene shuffling artifacts.